Abstract

1. Two ligand binding alpha subunits, alpha1 and alpha2, of the human (H) glycine receptor (GlyR) are involved at inhibitory synapses in the adult and neonatal spinal cord, respectively. The ability of homomeric alphaH1 and alphaH2 GlyRs to be activated by glycine, taurine and GABA was studied in Xenopus oocytes or in the human embryonic kidney HEK-293 cell line. 2. In outside-out patches from HEK cells, glycine, taurine and GABA activated both GlyRs with the same main unitary conductance, i.e. 85 +/- 3 pS (n = 6) for alphaH1, and 95 +/- 5 pS (n = 4) for alphaH2. 3. The sensitivity of both alphaH1 and alphaH2 GlyRs to glycine was highly variable. In Xenopus oocytes the EC50 for glycine (EC50gly) was between 25 and 280 microM for alphaH1 (n = 44) and between 46 and 541 microM for alphaH2 (n = 52). For both receptors, the highest EC50gly values were found on cells with low maximal glycine responses. 4. The actions of taurine and GABA were dependent on the EC50gly: (i) their EC50 values were linearly correlated to EC50gly, with EC50tau approximately 10 EC50gly and EC50GABA approximately 500-800 EC50gly; (ii) they could act either as full or weak agonists depending on the EC50gly. 5. The Hill coefficient (n(H)) of glycine remained stable regardless of the EC50gly whereas n(H) for taurine decreased with increasing EC50tau. 6. The degree of desensitization, evaluated by fast application of saturating concentrations of agonist on outside-out patches from Xenopus oocytes, was similar for glycine and taurine on both GlyRs and did not exceed 50 %. 7. Our data concerning the variations of EC50gly and the subsequent behaviour of taurine and GABA could be qualitatively described by the simple del Castillo-Katz scheme, assuming that the agonist gating constant varies whereas the binding constants are stable. However, the stability of the Hill coefficient for glycine was not explained by this model, suggesting that other mechanisms are involved in the modulation of EC50.

Variability of the EC50 for glycine of human homomeric αH1 and αH2 GlyRs

A, left: glycine-induced whole-cell currents from the most (upper traces) and the least (lower traces) sensitive oocytes expressing αH1 GlyRs recorded in this study. Concentrations are indicated above each trace. A, right: concentration dependencies for the same cells fitted by the Hill equation. The lowest EC50gly of αH1 was 25 μm, the highest 280 μm. B, similar presentation as in A for data recorded from oocytes expressing αH2 GlyRs. The lowest and highest EC50gly were 46 and 541 μm, respectively.

A, left: patch containing αH1 GlyRs. Currents induced by the three agonists at the concentrations indicated above each trace recorded at Vh = -70 mV. Note that currents obtained with glycine, taurine and GABA had similar amplitudes. A, right: I-V relationship of single-channel currents induced on the same patch by the three agonists. Only the main conductance for each is presented. At hyperpolarizing potentials, the conductance slope was 86 pS. B, same presentation as in A but for αH2 GlyRs. Typical traces (Vh = -60 mV) of agonist-induced currents are shown on the left. The I-V relationships from this patch (right) indicate that the three agonists activated a similar main conductance of 97 pS.

A and B, maximal currents induced by saturating concentrations of glycine, taurine and GABA and dose-response curves from two Xenopus oocytes expressing typical highly sensitive αH1 (A) and αH2 (B) subunits. EC50 values of the three tested agonists are indicated. The relative maximum responses of taurine and GABA, determined as Imaxagonist/Imaxgly from the illustrated traces, were 90 % and 69 % for the αH1 GlyR and 86 % and 73 % for the αH2 GlyR, respectively. Holding potentials were -70 mV.

Relative maximum responses of taurine and GABA depend on the EC50gly of the GlyRs

The relative maximum responses of taurine (determined as Imaxtau/Imaxgly; A) and of GABA (ImaxGABA/Imaxgly; B) from oocytes expressing αH1 or αH2 GlyRs were plotted against the EC50gly. Note the similarities between the two GlyRs.

Estimation of the desensitization from outside-out currents activated by glycine, taurine and GABA

A and B, currents induced by high concentrations of glycine (1 mm), taurine (10 or 30 mm) and GABA (50 or 100 mm) applied on outside-out patches from Xenopus oocytes using a fast perfusion system. Vh = -20 mV. C, responses of taurine and GABA normalized to the Igly. For each patch, the ratio Iagonist/Igly was measured either at the peak (□) or at the end of the pulse (I500 ms, ▪). Results are given ±s.d. Asterisk indicates a statistical difference between the mean ratio at the peak and at 500 ms (P < 0.001, t test). D, degree of desensitization of the currents induced by the three agonists, given by the ratio of the amplitude at 500 ms (I500 ms) and the current at the peak (Ipeak). Results are given ±s.d. Only patches on which all three agonists could be tested were included in the analysis.

Comparison of the relative maximum responses of taurine on wild-type αH1 GlyRs and on mutant αH1 GlyRs

The ratio Imaxtau/Imaxgly obtained in this study on αH1 GlyRs (•) are compared with those observed by , ○) on mutant and wild-type αH1 GlyRs expressed in transfected HEK cells and with those from , ; □) on wild-type and mutated αH1 GlyRs expressed in Xenopus oocytes. Data from mutants I244A and W243A which exhibit an accelerated desensitization () were not included in the graph.

Predictions from the del Castillo-Katz scheme when the binding constant (KA) remains stable while the gating constant (E) varies

A, dependence of Pmaxgly on c50gly. c50 is defined as the EC50 normalized to its binding constant and Pmax is the maximum fraction of open channel. Note the similar shape of this curve and the experimental results presented in ., distribution of the c50 for taurine and GABA as a function of the c50 for glycine. Considering our result (), the efficacies of taurine and GABA were fixed here and in C to Etau = 0.1 ×Egly and EGABA = 0.002 ×Egly. Note the similarity with ., maximum fraction of open channel by taurine or by GABA relative to Pmaxgly decline with increasing c50gly. Note the similarities with .